Method for treating a patient with neoplasla by treatment with a platinum coordination complex

ABSTRACT

This invention provides a method for treating a patient with neoplasia by an adjuvant therapy that includes treatment with an antineoplastic platinum coordination complex.

BACKGROUND OF THE INVENTION

[0001] Virtually all of the many antineoplastic drugs that are currentlyused in the treatment of cancer have very serious and harmful sideeffects. This is because cancer is generally treated with medicationsthat interfere with the growth of rapidly dividing cells. Suchmedications can inhibit the growth of the cancer cells, but they almostalways also inhibit the growth of normal cells that divide rapidly inthe body. Some of the normal tissues that divide very rapidly includebone marrow (which produces blood cells), hair follicles, and intestinalepithelium. The usefulness of virtually all antineoplastic drugs isseverely limited by the damage they cause to these normal tissues.

[0002] This invention relates to methods for treating neoplasia usingboth an antineoplastic platinum coordination complex (a commonchemotherapeutic) and a cyclic GMP (cGMP)-specific phosphodiesterase(PDE) inhibitor to reduce the side effects or increase the efficacy oftreatment with an antineoplastic platinum coordination complex. Undercurrent practices, platinum complexes (e.g., cisplatin and carboplatin)are typically used to treat certain cancers, particularly ovarian andtesticular cancers.

[0003] Cisplatin (cis-diaminedichloroplatinum) is a heavy metal complexwith a central platinum atom surrounded by two ammonia molecules and twochlorine atoms in the cis position. Cisplatin is also known by the tradename Platinol.

[0004] Cisplatin is typically used as a secondary therapy in combinationwith other chemotherapeutic agents for metastatic testicular tumors andmetastatic ovarian tumors in patients who have already receivedappropriate surgical or radiotherapeutic treatment. Cisplatin is alsoused as a single agent in treating patients with transitional cellbladder cancer which is not suited for surgical or radiotherapeutictreatment. Cisplatin has been used in treating epithelial malignanciesas well as cancers of the head and neck, the esophagus and the lung.

[0005] Cisplatin appears to enter cells by diffusion. The chlorine atomsof cisplatin are subject to chemical displacement by nucleophiles, suchas water or sulfhydryl groups. The activated species of the drug reactswith nucleic acids and proteins. Platinum complexes can react with DNA,forming both intrastrand and interstrand crosslinks, which inhibit DNAreplication and RNA transcription and can lead to breaks and miscoding.The platinum from cisplatin also becomes bound to several plasmaproteins including albumin, transferrin, and gamma globulin which mayinterfere with a number of cellular functions.

[0006] The major dose-limiting toxicity of cisplatin is cumulative renalinsufficiency which has been associated with renal tubular damage. Renaltoxicity becomes more prolonged and more severe with repeated cisplatintreatments. Electrolyte disturbances are often secondary effects ofrenal damage. Hydration and diuresis are used to reduce renal toxicity,but renal damage often occurs even if these measures are taken.

[0007] Myelosuppression is another dose-related toxicity of cisplatintreatment, characterized by a decrease in the levels of leukocytes andplatelets. Leukocytes are white blood cells which fight off infection,and platelets are necessary for proper blood clotting. Anemia is anotherside effect of treatment with cisplatin.

[0008] Toxic reactions in the ears, or otoxicity is another effect ofcisplatin treatment. This can be manifested by tinnitus, or noises suchas ringing or whistling in the ears, loss of high frequency hearing, andoccasionally deafness. It is unclear whether cisplatin-relatedototoxicity is reversible.

[0009] Other side effects of cisplatin include gastrointestinal effectssuch as nausea and vomiting which occur in almost all patients treatedwith cisplatin. Anaphylactic-like reactions may occur shortly afteradministration of the drug.

[0010] Cisplatin is a member of the family of platinum coordinationcomplexes. There are numerous derivatives of cisplatinum includingcarboplatin and oxaliplatin. Carboplatin (Paraplatin), like cisplatin,is thought to produce interstrand DNA cross-links. It is currently usedin the treatment of patients with ovarian cancer that has recurred afterchemotherapy. Clinically, there is less nephrotoxicity with carboplatinthan with cisplatin, and the dose-limiting toxicity with carboplatin ismyelosupression, primarily as thrombocytopenia, or a decrease in the ofplatelets circulating in the blood.

SUMMARY OF THE INVENTION

[0011] This invention relates to an improved method of cancer therapythat involves treating a patient with both an antineoplastic platinumcoordination complex (i.e., a cisplatin derivative, which includes bothcisplatin and derivatives thereof such as carboplatin) and acGMP-specific phosphodiesterase (PDE) inhibitor. The specific PDEinhibitors useful for this invention are compounds that inhibit bothPDE5 and the new cGMP-specific PDE described below. The novel cGMP-PDEis fully described by Liu, et al., in the copending U.S. patentapplication Ser. No. 09/173,375 (Case No. P-143), A Novel CyclicGMP-Specific Phosphodiesterase And Methods For Using Same InPharmaceutical Screening For Identifying Compounds For Inhibition OfNeoplastic Lesions. (For general PDE background, see, Beavo, J. A.(1995) Cyclic nucleotide phosphodiesterases: functional implications ofmultiple isoforms. Physiological Reviews 75:725-747; web site<http://weber.u.washington.edu/˜pde/pde.html>(November 1998)).

[0012] In this invention, the cGMP-specific PDE inhibitor can be used incombination with an antineoplastic platinum coordination complex in twoways. The first is a lower dosage methodology in which the traditionallyrecommended dose range of the cisplatin derivative is decreased whileits therapeutic effects are maintained and its side effects areattenuated. The second is a higher dosage methodology that utilizes thetraditionally recommended dose range for the cisplatin derivative andimproves its activity without increasing its side effects. With eachmethodology, the cisplatin derivative is administered simultaneouslywith or in succession with an appropriate cGMP-specific PDE inhibitor.

[0013] In the low dose regime, a cisplatin derivative is administered atdoses less than about 75 mg/m². In the high dose regime, a cisplatinderivative is administered at doses between about 75 and 100 mg/m².

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a graph of the cGMP activities of the cGMPphosphodiesterases obtained from SW-480 neoplastic cells, as assayedfrom a the eluent from a DEAE-Trisacryl M column.

[0015]FIG. 2 is a graph of cGMP activities of the reloaded cGMPphosphodiesterases obtained from SW-480 neoplastic cells, as assayedfrom a the eluent from a DEAE-Trisacryl M column.

[0016]FIG. 3 is a graph of the kinetic behavior of the novel PDE.

[0017]FIG. 4 illustrates the inhibitory effects of sulindac sulfide andexisulind on PDE4 and PDE5 purified from cultured tumor cells.

[0018]FIG. 5 illustrates the effects of sulindac sulfide on cyclicnucleotide levels in HT-29 cells.

[0019]FIG. 6 illustrates the phosphodiesterase inhibitory activity ofCompound B.

[0020]FIG. 7 illustrates the phosphodiesterase inhibitory activity ofCompound E.

[0021]FIG. 8 illustrates the effects of sulindac sulfide and exisulindon tumor cell growth.

[0022]FIG. 9 illustrates the growth inhibitory and apoptosis-inducingactivity of sulindac sulfide and control (DMSO).

[0023]FIG. 10 illustrates the growth inhibitory activity of compound E.

[0024]FIG. 11 illustrates the effects of sulindac sulfide and exisulindon apoptosis and necrosis of HT-29 cells.

[0025]FIG. 12 illustrates the effects of sulindac sulfide and exisulindon HT-29 cell growth inhibition and apoptosis induction as determined byDNA fragmentation.

[0026]FIG. 13 illustrates the apoptosis inducing properties of CompoundE.

[0027]FIG. 14 illustrates the apoptosis inducing properties of CompoundB.

[0028]FIG. 15 illustrates the inhibition of pre-malignant, neoplasticlesions in mouse mammary gland organ culture by sulindac metabolites.

[0029]FIG. 16 illustrates the effects of carboplatin and exisulind(FGN-1) on tumor cell growth at 25 μM exisulind.

[0030]FIG. 17 illustrates the effects of carboplatin and exisulind(FGN-1) on tumor cell growth at 50 μM exisulind.

[0031]FIG. 18 illustrates the effects of carboplatin and exisulind(FGN-1) on tumor cell growth at 100 μM exisulind.

[0032]FIG. 19 illustrates the effects of carboplatin and exisulind(FGN-1) on tumor cell growth at 200 μM exisulind.

[0033]FIG. 20 illustrates the effects of carboplatin and exisulind(FGN-1) on tumor cell growth at 400 μM exisulind.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] As discussed in greater detail below, the inhibition ofcGMP-specific PDEs can induce apoptosis in neoplastic cells. Cisplatinderivatives are currently used to treat neoplasias, particularly ovarianand testicular cancers. The combination of these two types of therapiescan produce an effect that neither can produce individually.

[0035] I. The Novel cGMP-Specific Phosphodiesterase

[0036] A new cyclic GMP-specific phosphodiesterase has been discoveredin neoplastic cells. Treatment of cells with a compound that inhibitsboth PDE5 and this novel cGMP-specific PDE leads to apoptosis of theneoplastic cells. In other words, the preferred cGMP-specific inhibitorsuseful in this invention, in combination with a cisplatin derivative arethose compounds that inhibit both PDE5 and this new PDE.

[0037] The new PDE is broadly characterized by

[0038] (a) cGMP specificity over cAMP;

[0039] (b) positive cooperative kinetic behavior in the presence of cGMPsubstrate;

[0040] (c) submicromolar affinity for cGMP; and

[0041] (d) insensitivity to incubation with purified cGMP-dependentprotein kinase.

[0042] As discussed below, this new cGMP-PDE is unique from theclassical PDE5. Kinetic data reveal that the new PDE has increased cGMPhydrolytic activity in the presence of increasing cGMP substrateconcentrations, unlike PDE5 which exhibits cGMP substrate saturation.The new cGMP-PDE is insensitive to incubation with cGMP-dependentprotein kinase (PKG), whereas PDE5 is phosphorylated by PKG.Additionally, the new cGMP-PDE is relatively insensitive to inhibitionwith the PDE5-specific inhibitors, zaprinast and E4021. Finally, the newcGMP-PDE activity can be separated from classical PDE5 activity byanion-exchange chromatography.

[0043] The new cGMP-PDE is not a member of any of the other previouslycharacterized PDE families. The new PDE does not hydrolyze cAMPsignificantly. Calcium (with or without calmodulin) failed to activateeither cAMP or cGMP hydrolysis activity, indicating that the novel PDEis not a CaM-PDE (PDE1). Additionally, cGMP failed to activate orinhibit cAMP hydrolysis, indicating that the new cGMP-PDE it is not acGMP-stimulated PDE (cGS-PDE or PDE2), because all known isoforms of thePDE2 family hydrolyze both cAMP and cGMP. Further, the new cGMP-PDE isinsensitive to a number of specific PDE inhibitors. It is relativelyinsensitive to vinpocetine (a CaM-PDE- or PDE1-specific inhibitor), toindolodan (a cGI-PDE- or PDE3-specific inhibitor), and to rolipram (acAMP-PDE- or PDE4-specific inhibitor). The data establish that the newPDE is not a member of one of the cAMP-hydrolyzing PDE families (PDE1,PDE2, PDE3, or PDE4).

[0044] PDE inhibitors that are useful for treating patients withneoplasia consistent with this invention should inhibit both PDE5 andthe new cGMP-PDE. A compound that inhibits both forms of cGMP-specificPDE is desirable because a compound that inhibits PDE5 but not the newPDE, does not by itself induce apoptosis. For example, zaprinast,sildenafil, and E4021 have been reported as potent inhibitors of PDE5.However, compared to PDE5, the new PDE is relatively insensitive tozaprinast, sildenafil, and E4021 (Table 1). And none of the three,zaprinast, sildenafil, or E4021, have been found to induce apoptosis(Table 6) or to inhibit cell growth in neoplastic cells (Tables 3 and4).

[0045] However, a number of PDE5 inhibitors have been found to induceapoptosis in neoplastic cells. Examples of such compounds are sulindacsulfide and Compound E. Sulindac sulfide and Compound E each inhibitPDE5 and the new cGMP-PDE with the same potency (Table 1). And bothsulindac sulfide and Compound E induce apoptosis in neoplastic cells(Table 6). Compounds that inhibit PDE5, but not the new cGMP-PDE, do notcause apoptosis in neoplastic cells. But compounds that inhibit bothPDE5 and the new cGMP-PDE, have been found to induce apoptosis inneoplastic cells.

[0046] A. Isolation of the Novel cGMP-Specific Phosphodiesterase

[0047] The novel cGMP-specific phosphodiesterase can be isolated fromhuman carcinoma cell lines (e.g. SW-480, a human colon cancer cell linethat originated from a moderately differentiated epithelialadenocarcinoma, available from the American Tissue Type Collection inRockville, Md., U.S.A.). The complete isolation of this new cGMP-PDE isdescribed in the copending application, Liu, et al., U.S. patentapplication Ser. No. ______ (Case No. P-143), A Novel CyclicGMP-Specific Phosphodiesterase And Methods For Using Same InPharmaceutical Screening For Identifying Compounds For Inhibition OfNeoplastic Lesions, which is incorporated herein by reference.

[0048] Briefly, to isolate the novel phosphodiesterase, SW-480 cells arecollected and homogenized. The homogenate is centrifuged, and thesupernatant is loaded onto a DEAE-Trisacryl M column. The loaded columnis then washed, and PDE activities are eluted with a linear gradient ofNaOAc. Fractions are collected and immediately assayed for cGMPhydrolysis activity. Cyclic nucleotide PDE activity of each fraction isdetermined using the modified two-step radioisotopic method of Thompsonet al. (Thompson W. J., et al., Adv Cyclic Nucleotide Res 10: 69-92,1979). There are two initial peaks of cGMP-PDE activity eluted from thecolumn, peak A and peak B (see FIG. 1). Peak A is PDE5, whereas peak Bis the new cGMP-PDE.

[0049] To further fractionate the cGMP hydrolytic activity of PDE5 andthe new cGMP-PDE, the fractions containing those activities are reloadedonto the DEAE-Trisacryl M column and eluted with a linear gradient ofNaOAc. Fractions are again immediately assayed for cGMP hydrolysisactivity, the results of which are presented in FIG. 2. As illustratedin FIG. 2, peak B, the novel PDE, shows enhanced activity withincreasing cGMP substrate concentration. Peak A, on the other hand,shows apparent substrate saturation with increasing concentrations ofcGMP.

[0050] B. cGMP-Specificity of PDE Peaks A and B

[0051] Each fraction from the DEAE column was also assayed forcGMP-hydrolysis activity (0.25 μM cGMP) in the presence or absence ofCa_(—, or Ca) _(—-CaM and/or EGTA and for cAMP ()0.25 μM cAMP)hydrolysis activity in the presence or absence of 5 μM cGMP. Neither PDEpeak A nor peak B (fractions 5-22; see FIG. 1) hydrolyzed cAMPsignificantly, establishing that neither was a member of a cAMPhydrolyzing family of PDEs (i.e. a PDE 1, 2, 3).

[0052]Ca_(—(with or without calmodulin) failed to activate either cAMP or cGMP hydrolysis activity of either peak A or B, and cGMP failed to activate or inhibit cAMP hydrolysis. Such results establish that peaks A and B constitute cGMP-specific PDEs but not PDE)1,PDE2, PDE3, or PDE4.

[0053] For PDE peak B, as discussed below, cyclic GMP activated the cGMPhydrolytic activity of the enzyme, but did not activate any cAMPhydrolytic activity. This reveals that PDE peak B—the novelphosphodiesterase—is not a cGMP-stimulated cyclic nucleotide PDE (“cGS”)or among the PDE2 family isoforms because the known isoforms of PDE2hydrolyze both cGMP and cAMP.

[0054] C. Peak A is a PDE5, but Peak B—a New cGMP-Specific PDE—is not

[0055] To characterize any PDE isoform, kinetic behavior and substratepreference should be assessed. Peak A showed typical “PDE5”characteristics. For example, the K_(m) of the enzyme for cGMP was 1.07μM, and Vmax was 0.16 nmol/min/mg. In addition, as discussed below,zaprinast (IC₅₀=1.37 μM), E4021 (IC₅₀=3 nM), and sildenafil inhibitedactivity of peak A. Further, zaprinast showed inhibition for cGMPhydrolysis activity of peak A, consistent with results reported in theliterature for PDE5.

[0056] PDE peak B showed considerably different kinetic properties ascompared to PDE peak A. For example, in Eadie-Hofstee plots of peak A,cyclic GMP hydrolysis shows a single line with negative slope withincreasing substrate concentrations, indicative of Michaelis-Mentenkinetic behavior. Peak B, however, shows the novel property for cGMPhydrolysis in the absence of cAMP of a decreasing (apparent K_(m)=8.4),then increasing slope (K_(m)<1) of Eadie-Hofstee plots with increasingcGMP substrate (see FIG. 3). This establishes peak B's submicromolaraffinity for cGMP (i.e., where K_(m)<1).

[0057] Consistent with the kinetic studies (i.e., FIG. 3) andpositive-cooperative kinetic behavior in the presence of cGMP substrate,was the increased cGMP hydrolytic activity in the presence of increasingconcentrations of cGMP substrate. This was discovered by comparing 0.25μM, 2 μM, and 5 μM concentrations of cGMP in the presence of PDE peak Bafter a second DEAE separation to rule out cAMP hydrolysis and to ruleout this new enzyme being a “classic” PDE5. Higher cGMP concentrationsevoked disproportionately greater cGMP hydrolysis with PDE peak B asshown in FIG. 2.

[0058] These observations suggest that cGMP binding to the peak B enzymecauses a conformational change in the enzyme.

[0059] D. Zaprinast- and Sildenafil-Insensitivity of PDE Peak B Relativeto Peak A, and Their Effects on Other PDE Inhibitors

[0060] Different PDE inhibitors were studied using twelve concentrationsof drug from 0.01 to 100 μM and substrate concentration of 0.25 μM³H-cGMP. IC₅₀ values were calculated with variable slope, sigmoidalcurve fits using Prism 2.01 (GraphPad). The results are shown inTable 1. While compounds E4021 and zaprinast inhibited peak A, (withhigh affinities) IC₅₀ values calculated against peak B are significantlyincreased (>50 fold). This confirms that peak A is a PDE5. These datafurther illustrate that the novel PDE is, for all practical purposes,zaprinast-insensitive and E4021-insensitive. TABLE 1 Comparison of PDEInhibitors Against Peak A and Peak B (cGMP Hydrolysis) Ratio (IC₅₀ PDEFamily IC₅₀ IC₅₀ Peak A/ Compound Inhibitor Peak A (μM) Peak B (μM) PeakB) E4021 5 0.003 8.4 0.0004 Zaprinast 5 1.4 >30 <0.05 Compound E 5 andothers 0.38 0.37 1.0 Sulindac 5 and others 50 50 1.0 sulfide Vinpocetine1 >100 >100 EHNA 2, 5 >100 3.7 Indolidan 3 31 >100 <0.31 Rolipram4 >100 >100 Sildenafil 5 .0003 >10 <.00003

[0061] By contrast, sulindac sulfide and Compound E competitivelyinhibit both peak A and peak B phosphodiesterases at the same potency(for Compound E, IC₅₀=0.38 μM for PDE peak A; IC₅₀=0.37 μM for PDE peakB).

[0062] There is significance for the treatment of neoplasia and theselection of useful compounds for such treatment in the fact that peak Bis zaprinast-insensitive whereas peaks A and B are both sensitive tosulindac sulfide and Compound E. Zaprinast, E4021, and sildenafil havebeen tested to ascertain whether they induce apoptosis or inhibit thegrowth of neoplastic cells, and the same has been done for Compound E.As explained below, zaprinast, sildenafil and E4021 do not havesignificant apoptosis-inducing (Table 6) or growth-inhibiting (Tables 3and 4) properties, whereas sulindac sulfide and Compound E are preciselythe opposite. In other words, the ability of a compound to inhibit bothPDE peaks A and B correlates with its ability to induce apoptosis inneoplastic cells, whereas if a compound, (e.g., zaprinast) hasspecificity for PDE peak A only, that compound will not induceapoptosis.

[0063] E. Insensitivity of PDE Peak B to Incubation with cGMP-DependentProtein Kinase

[0064] Further differences between PDE peaks A and B were observed intheir respective cGMP-hydrolytic activities in the presence of varyingconcentrations of cGMP-dependent protein kinase (PKG, whichphosphorylates typical PDE5). Specifically, peak A and peak B fractionswere incubated with different concentrations of protein kinase G at 30°C. for 30 minutes. Cyclic GMP hydrolysis of both peaks was assayed afterphosphorylation was attempted. Consistent with previously publishedinformation about PDE5, peak A showed increasing cGMP hydrolysisactivity in response to protein kinase G incubation, indicating thatpeak A was phosphorylated. Peak B was unchanged, however (i.e., was notphosphorylated and was insensitive to incubation with cGMP-dependentprotein kinase). These data are consistent with peak A being a PDE5family isoform and peak B being a novel cGMP-PDE.

[0065] II. Selecting a cGMP-Specific Phosphodiesterase Inhibitor for usein this Invention

[0066] Cancer and precancer may be thought of as diseases that involveunregulated cell growth. Cell growth involves a number of differentfactors. One factor is how rapidly cells proliferate, and anotherinvolves how rapidly cells die. Cells can die either by necrosis orapoptosis depending on the type of environmental stimuli. Celldifferentiation is yet another factor that influences tumor growthkinetics. Resolving which of the many aspects of cell growth is affectedby a test compound is important to the discovery of a relevant targetfor pharmaceutical therapy. Assays based on this technology can becombined with other tests to determine which compounds have growthinhibiting and proapoptotic activity.

[0067] In this invention, particular cGMP-specific PDE inhibitors areselected for use in combination with a cisplatin derivative to treatneoplasia, especially ovarian and testicular cancers, in one of severalways. As indicated above, preferred PDE inhibitors are those thatinhibit the activities of both PDE5 and the new cGMP-PDE. A compound canbe selected for use in this invention by evaluating its effect on thecGMP hydrolytic activity on a mixture of the two enzymes (i.e., amixture of peaks A and B) isolated from a tumor cell line.Alternatively, a compound can be selected by evaluating the compound'seffect on cyclic nucleotide levels in whole neoplastic cells before andafter exposure of the cells to the compound of interest. Still anotheralternative is to test a compound of interest against the two PDEsseparately, i.e., by physically separating each activity from a tumorcell line (or by using recombinant versions of each enzyme) and testingthe inhibitory action of the compound against each enzyme individually.With any of the above approaches, an appropriate PDE inhibitor can beselected for use in combination with a cisolatin derivative.

[0068] A. Phosphodiesterase Enzyme Assay

[0069] Phosphodiesterase activity (whether in a mixture or separately)can be determined using methods known in the art, such as a method usinga radioactively labeled form of cGMP as a substrate for the hydrolysisreaction. Cyclic GMP labeled with tritium (³H-cGMP) is used as thesubstrate for the PDE enzymes. (Thompson, W. J., Teraski, W. L.,Epstein, P. M., Strada, S. J., Advances in Cyclic Nucleotide Research,10:69-92, 1979, which is incorporated herein by reference). In thisassay, cGMP-PDE activity is determined by quantifying the amount of cGMPsubstrate that is hydrolyzed either in the presence or absence of thetest compound).

[0070] In brief, a solution of defined substrate ³H-cGMP specificactivity is mixed with the drug to be tested. The mixture is incubatedwith isolated PDE activity (either a single PDE or a mixture of PDEactivities). The degree of phosphodiesterase inhibition is determined bycalculating the amount of radioactivity released in drug-treatedreactions and comparing those against a control sample (a reactionmixture lacking the tested compound but with the drug solvent).

[0071] B. Cyclic Nucleotide Measurements

[0072] Alternatively, the ability of a compound to inhibit cGMP-PDEactivity is reflected by an increase in the levels of cGMP in neoplasticcells exposed to the test compound. The amount of PDE activity can bedetermined by assaying for the amount of cyclic GMP in the extract oftreated cells using a radioimmunoassay (RIA). In this procedure, aneoplastic cell line is incubated with a test compound. After about 24to 48 hours, the cells are solubilized, and cyclic GMP is purified fromthe cell extracts. The cGMP is acetylated according to publishedprocedures, such as using acetic anhydride in triethylamine, (Steiner,A. L., Parker, C. W., Kipnis, D. M., J. Biol. Chem., 247(4):1106-13,1971, which is incorporated herein by reference). The acetylated cGMP isquantitated using radioimmunoassay procedures (Harper, J., Brooker, G.,Advances in Nucleotide Research, 10:1-33, 1979, which is incorporatedherein by reference).

[0073] In addition to observing increases in the content of cGMP inneoplastic cells as a result of incubation with certain test compounds,decreases in the content of cAMP have also been observed. It has beenobserved that a compound which is useful in the practice of thisinvention (i.e., one that selectively induces apoptosis in neoplasticcells, but not substantially in normal cells) follows a time courseconsistent with cGMP-specific PDE inhibition. Initially, the result isan increased cGMP content within minutes, and secondarily, there is adecreased cAMP content within 24 hours. The intracellular targets ofthese drug actions are being studied further, but current data supportthe concept that the initial rise in cGMP content and the subsequentfall in cAMP content precede apoptosis in neoplastic cells exposed totest compounds useful in this invention. To determine the cyclic AMPcontent in cell extracts, radioimmunoassay techniques similar to thosedescribed above for cGMP are used.

[0074] The change in the ratio of the two cyclic nucleotides may be amore accurate tool for evaluating cGMP-specific phosphodiesteraseinhibition activity of test compounds, rather than measuring only theabsolute value of cGMP, only the level of cGMP hydrolysis, or onlycGMP-specific phosphodiesterase inhibition. In neoplastic cells nottreated with anti-neoplastic compounds, the ratio of cGMP content/cAMPcontent is in the 0.03-0.05 range (i.e., 300-500 fmol/mg protein cGMPcontent over 6000-8000 fmol/mg protein cAMP content). After exposure todesirable anti-neoplastic compounds, that ratio increases several fold(preferably at least about a three-fold increase) as the result of aninitial increase in cyclic GMP and the later decrease in cyclic AMP.

[0075] Specifically, it has been observed that particularly desirablecompounds achieve an initial increase in cGMP content in treatedneoplastic cells to a level of cGMP greater than about 500 fmol/mgprotein. In addition, particularly desirable compounds cause the laterdecrease in cAMP content in treated neoplastic cells to a level of cAMPless than about 4000 fmol/mg protein.

[0076] Verification of the cyclic nucleotide content may be obtained bydetermining the turnover or accumulation of cyclic nucleotides in intactcells. To measure the levels of cAMP in intact cells, ³H-adenineprelabeling is used according to published procedures (Whalin M. E., R.L. Garrett Jr., W. J. Thompson, and S. J. Strada, “Correlation ofcell-free brain cyclic nucleotide phosphodiesterase activities to cyclicAMP decay in intact brain slices”, Sec. Mess. and Phos. ProteinResearch, 12:311-325, 1989, which is incorporated herein by reference).The procedure measures flux of labeled ATP to cyclic AMP and can be usedto estimate intact cell adenylate cyclase or cyclic nucleotidephosphodiesterase activities depending upon the specific protocol.Cyclic GMP accumulation was too low to be studied with intact cellprelabeling according to published procedures (Reynolds. P. E., S. J.Strada and W. J. Thompson, “Cyclic GMP accumulation in pulmonarymicrovascular endothelial cells measured by intact cell prelabeling,”Life Sci., 60:909-918, 1997, which is incorporated herein by reference).

[0077] C. Tissue Sample Assay

[0078] The cGMP-specific PDE inhibitory activity of a test compound canalso be determined from a tissue sample. Tissue biopsies from humans ortissues from anesthetized animals are collected from subjects exposed tothe test compound. Briefly, a sample of tissue is homogenized and aknown amount of the homogenate is removed for protein analysis. From theremaining homogenate, the protein is allowed to precipitate. Next, thehomogenate is centrifuged and both the supernatant and the pellet arerecovered. The supernatant is assayed for the amount of cyclicnucleotides present using RLk procedures as described above.

[0079] D. Experimental Results

[0080] 1. Introduction

[0081] The amount of cGMP-specific inhibition is determined by comparingthe activity of the cGMP-specific PDEs in the presence and absence ofthe test compound. Inhibition of cGMP-PDE activity is indicative thatthe compound is useful for treating neoplasia in combination with acisplatin derivative. Significant inhibitory activity, greater than thatof the benchmark, exisulind, and preferably greater than 50% at aconcentration of 10 μM or below, is indicative that a compound should befurther evaluated for antineoplastic properties. The term “exisulind”means (Z)-5-fluoro-2-methyl-1-[[4-(methylsulfonyl)phenyl]methylene]indene-3-yl acetic acid or a salt thereof. (See, Pamukcu andBrendel, U.S. Pat. No. 5,401,774.)

[0082] 2. cGMP-PDE Inhibition Assay

[0083] Reference compounds and test compounds were analyzed for theircGMP-PDE inhibitory activity in accordance with the protocol for theassay described supra. FIG. 4 shows the effect of various concentrationsof sulindac sulfide and exisulind on either PDE4 or cGMP-PDE activitypurified from human colon HT-29 cultured tumor cells, as describedpreviously (W. J. Thompson et al., supra). The IC₅₀ value of sulindacsulfide for inhibition of PDE4 was 41 μM, and for inhibition of cGMP-PDEwas 17 μM. The IC₅₀ value of exisulind for inhibition of PDE4 was 181μM, and for inhibition of cGMP-PDE was 56 μM. These data show that bothsulindac sulfide and exisulind inhibit phosphodiesterase activity. Bothcompounds show selectivity for the cGMP-PDE isoenzyme forms over PDE4isoforms.

[0084]FIG. 5 shows the effects of sulindac sulfide on either cGMP orcAMP production as determined in cultured HT-29 cells in accordance withthe assay described, supra. HT-29 cells were treated with sulindacsulfide for 30 minutes and cGMP or cAMP was measured by conventionalradioimmunoassay method. As indicated, sulindac sulfide increased thelevels of cGMP by greater than 50% with an EC₅₀ value of 7.3 μM (FIG.5A, top). Levels of cAMP were unaffected by treatment, although a knownPDE4 inhibitor, rolipram, increased cAMP levels (FIG. 5B, bottom). Thedata demonstrate the pharmacological significance of inhibitingcGMP-PDE, relative to PDE4.

[0085]FIG. 6 shows the effect of the indicated dose of test Compound B,described below, on either cGMP-PDE or PDE4 isozymes ofphosphodiesterase. The calculated IC₅₀ value was 18 μM for cGMP-PDE and58 μM for PDE4.

[0086]FIG. 7 shows the effect of the indicated dose of test Compound E.described below, on either PDE4 or cGMP-PDE. The calculated IC₅₀ valuewas 0.08 μM for cGMP-PDE and greater than 25 μM for PDE4.

[0087] Compounds

[0088] A number of compounds were examined in the various protocols andscreened for potential use in treating neoplasia. The results of thesetests are reported below. The test compounds are hereinafter designatedby a letter code that corresponds to the following:

[0089] A—rac-threo-(E)-1-(N,N′-diethylaminoethanethio)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;

[0090] B—(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-aceticacid;

[0091] C—(Z)-5-Fluoro-2-methyl-1-(p-chlorobenzylidene)-3-acetic acid;

[0092]D—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-N-acetylcysteine;

[0093]E—(Z)-5-Fluoro-2-methyl-1-(3,4,5-trimethoxybenzylidene)-3-indenylacetamide,N-benzyl;

[0094]F—(Z)-5-Fluoro-2-methyl-1-(p-methylsulfonylbenzylidene)-3-indenylacetamide,N,N′-dicyclohexyl;

[0095]G—ribo-(E)-1-Triazolo-[2′,3′:1″,3″]-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-indan;and

[0096]H—rac-(E)-1-(butan-1′,4′-olido)-[3′,4′:1,2]-6-fluoro-2-methyl-3-(p-methylsulfonylbenzylidene)-1S-indanyl-glutathione).TABLE 2 cGMP PDE Inhibitory Activity Among a Series of CompoundsReference compounds % Inhibition at 10 μM Indomethacin 34 MY5445 86Sulindac sulfide 97 Exisulind 39 Test compounds % Inhibition at 100 μM A<25 B <25 C <25 D 36 E 75

[0097] The above compounds in Table 2 were evaluated for PDE inhibitoryactivity in HT-29 cells, as described in the protocol, supra. Of thecompounds that did not inhibit COX, only Compound E was found to causegreater than 50% inhibition at 10 μM. As noted in FIG. 6, Compound Bshowed inhibition of greater than 50% at a dose of 20 μM. Therefore,depending on the dosage level used in a single dose test, some compoundsmay be screened out that otherwise may be active at slightly higherdosages. The dosage used is subjective and may be lowered to identifyeven more potent compounds after active compounds are found at certainconcentration levels.

[0098] III. Determining whether a Compound Reduces the Number of TumorCells

[0099] In an alternate embodiment, the preferred cGMP-specificinhibitors useful in the practice of this invention are selected byfurther determining whether the compound reduces the growth of tumorcells in vitro. Various cell lines can be used depending on the tissueto be tested. For example, these cell lines include: SW-480—colonicadenocarcinoma; HT-29—colonic adenocarcinoma; A-427—lung adenocarcinoma;MCF-7—breast adenocarcinoma; UACC-375—melanoma line; and DU145—prostratecarcinoma. Cytotoxicity data obtained using these cell lines areindicative of an inhibitory effect on neoplastic lesions. These celllines are well characterized, and are used by the United States NationalCancer Institute in their screening program for new anti-cancer drugs.

[0100] A. Tumor Inhibition in the HT-29 Cell Line

[0101] A compound's ability to inhibit tumor cell growth can be measuredusing the HT-29 human colon carcinoma cell line obtained from ATCC(Bethesda, Md.). HT-29 cells have previously been characterized as arelevant colon tumor cell culture model (Fogh, J., and Trempe, G. In:Human Tumor Cells in Vitro, J. Fogh (ed.), Plenum Press, New York, pp.115-159, 1975). Briefly, after being grown in culture, HT-29 cells arefixed by the addition of cold trichloroacetic acid. Protein levels aremeasured using the sulforhodamine B (SRB) colorimetric protein stainassay as previously described by Skehan, P., Storeng, R., Scudiero, D.,Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney,S., and Boyd, M. R., “New Colorimetric Assay For Anticancer-DrugScreening,” J. Natl. Cancer Inst. 82: 1107-1112, 1990, which isincorporated herein by reference.

[0102] In addition to the SRB assay, a number of other methods areavailable to measure growth inhibition and could be substituted for theSRB assay. These methods include counting viable cells following trypanblue staining, labeling cells capable of DNA synthesis with BrdU orradiolabeled thymidine, neutral red staining of viable cells, or MTTstaining of viable cells.

[0103] B. Experimental Results

[0104] 1. Introduction

[0105] Significant tumor cell growth inhibition, greater than about 50%at a dose of 100 μM or below is further indicative that the compound isuseful for treating neoplastic lesions. Preferably, an IC₅₀ value isdetermined and used for comparative purposes. This value is theconcentration of drug needed to inhibit tumor cell growth by 50%relative to the control. Preferably, the IC₅₀ value should be less than100 μM for the compound to be considered useful for treating neoplasticlesions in combination with a cisplatin derivative according to themethod of this invention.

[0106] 2. Growth Inhibition Assay

[0107] Reference compounds and test compounds were analyzed for theircGMP-PDE inhibitory activity in accordance with the protocol for theassay, supra. FIG. 8 shows the inhibitory effect of variousconcentrations of sulindac sulfide and exisulind on the growth of HT-29cells. HT-29 cells were treated for six days with various doses ofexisulind (triangles) or sulindac sulfide (squares) as indicated. Cellnumber was measured by a sulforhodamine assay as previously described(Piazza et al., Cancer Research, 55: 3110-3116, 1995). The IC₅₀ valuefor sulindac sulfide was approximately 45 μM and for exisulind wasapproximately 200 μM. The data show that both sulindac sulfide andexisulind are capable of inhibiting tumor cell growth.

[0108]FIG. 9 shows the growth inhibitory and apoptosis-inducing activityof sulindac sulfide. A time course experiment is shown involving HT-29cells treated with either vehicle, 0.1% DMSO (open symbols) or sulindacsulfide, 120 μM (closed symbols). Growth inhibition (FIG. 9A, top) wasmeasured by counting viable cells after trypan blue staining. Apoptosis(FIG. 9B, bottom) was measured by morphological determination followingstaining with acridine orange and ethidium bromide as describedpreviously (Duke and Cohen, In: Current Protocols in Immunology,3.17.1-3.17.16, New York, John Wiley and Sons, 1992). The datademonstrate that sulindac sulfide is capable of inhibiting tumor cellgrowth and that the effect is accompanied by an increase in apoptosis.All data were collected from the same experiment.

[0109]FIG. 10 shows the growth inhibitory activity of test Compound E.HT-29 colon adenocarcinoma cells were treated with the indicatedconcentration of Compound E for six days and cell number was determinedby the SRB assay. The calculated IC₅₀ value was 0.04 μM. TABLE 3 GrowthInhibitory Activity Among a Series of Compounds Reference compounds %Inhibition at 100 μM Indomethacin 75 MY5445 88 Sulindac sulfide 88Exisulind <50 E4021 <50 sildenafil <50 zaprinast <50 Test compounds %Inhibition at 100 μM A 68 B 77 C 80 D 78 E 62

[0110] In accordance with the screening protocol, supra, Compounds Athrough E were tested for growth inhibitory activity, as reported inTable 3 above. All the test compounds showed activity exceeding thebenchmark exisulind at a 100 μM single dose test.

[0111] The growth inhibitory activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 4 below. HT-29cell were treated for 6 days with various inhibitors ofphosphodiesterase. Cell growth was determined by the SRB assaydescribed, supra. The data below taken with those above show thatinhibitors of the cGMP-specific PDE activity were effective forinibiting tumor cell growth. TABLE 4 Growth Inhibitory Data for PDEInhibitors Growth inhibition Inhibitor Reported Selectivity (IC₅₀, μM)8-methoxy-IBMX PDE1 >200 μM Milrinone PDE3 >200 μM RO-20-1724 PDE4 >200μM MY5445 PDE5 5 μM IBMX Non-selective >100 μM Zaprinast PDE5 >100 μMSildenafil PDE5 >100 μM E4021 PDE5 >100 μM

[0112] To show the effectiveness of cGMP-specific PDE inhibition onvarious forms of neoplasia, compounds were tested on numerous celllines. The effects of sulindac sulfide and exisulind on various celllines was determined. The data are shown in Table 5 below. The IC₅₀values were determined by the SRB assay. The data show the effectivenessof these compounds on a broad range of neoplasias, with effectiveness atcomparable dose range. Therefore, compounds selected for cGMP-specificPDE inhibition in combination with a cisplatin derivative should beuseful for treating neoplasia, in particular ovarian and testicularcancers. TABLE 5 Growth Inhibitory Data of Various Cell Lines IC₅₀ (μM)*Cell Type/ Sulindac Com- Tissue specificity sulfide Exisulind pound EHT-29, Colon 60 120 0.10 HCT116, Colon 45 90 MCF7/S, Breast 30 90UACC375, Melanoma 50 100 A-427, Lung 90 130 Bronchial Epithelial Cells30 90 NRK, Kidney (non ras-transformed) 50 180 KNRK, Kidney (rastransformed) 60 240 Human Prostate Carcinoma PC3 82 0.90 Colo 205 1.62DU-145 0.10 HCT-15 0.60 MDA-MB-231 0.08 MDA-MB-435 0.04

[0113] IV. Determining Whether a Compound Induces Apoptosis

[0114] In a second alternate embodiment, preferably, the cGMP-specificPDE inhibitors useful in combination with a cisplatin derivative in thepractice of this invention induce apoptosis in cultures of tumor cells.

[0115] Two distinct forms of cell death may be described bymorphological and biochemical criteria: necrosis and apoptosis. Necrosisis accompanied by increased permeability of the plasma membrane; thecells swell and the plasma membrane ruptures within minutes. Apoptosisis characterized by membrane blebbing, condensation of cytoplasm, andthe activation of endogenous endonucleases.

[0116] Apoptosis occurs naturally during normal tissue turnover andduring embryonic development of organs and limbs. Apoptosis also isinduced by cytotoxic T-lymphocytes and natural killer cells, by ionizingradiation, and by certain chemotherapeutic drugs. Inappropriateregulation of apoptosis is thought to play an important role in manypathological conditions including cancer, AIDS, Alzheimer's disease,etc. Cyclic GMP-specific PDE inhibitors useful in this invention can beselected based on their ability to induce apoptosis in cultured tumorcells maintained under conditions as described above.

[0117] Treatment of cells with test compounds involves either pre- orpost-confluent cultures and treatment for two to seven days at variousconcentrations of the compound in question. Apoptotic cells are measuredby combining both the attached and “floating” compartments of thecultures. The protocol for treating tumor cell cultures with sulindacand related compounds to obtain a significant amount of apoptosis hasbeen described in the literature. (See, Piazza, G. A., et al., CancerResearch, 55:3110-16, 1995, which is incorporated herein by reference).The novel features include collecting both floating and attached cells,identification of the optimal treatment times and dose range forobserving apoptosis, and identification of optimal cell cultureconditions.

[0118] A. Analysis of Apoptosis by Morphological Observation

[0119] Following treatment with a test compound, cultures can be assayedfor apoptosis and necrosis by fluorescent microscopy following labelingwith acridine orange and ethidium bromide. The method for measuringapoptotic cell number has previously been described by Duke & Cohen,“Morphological And Biochemical Assays Of Apoptosis,” Current ProtocolsIn Immunology, Coligan et al., eds., 3.17.1-3.17.16 (1992, which 5sincorporated herein by reference).

[0120] For example, floating and attached cells can be collected, andaliquots of cells can be centrifuged. The cell pellet can then beresuspended in media and a dye mixture containing acridine orange andethidium bromide. The mixture can then be examined microscopically formorphological features of apoptosis.

[0121] B. Analysis of Apoptosis by DNA Fragmentation

[0122] Apoptosis can also be quantified by measuring an increase in DNAfragmentation in cells which have been treated with test compounds.Commercial photometric EIA for the quantitative in vitro determinationof cytoplasmic histone-associated-DNA-fragments (mono- andoligonucleosomes) are available (Cell Death Detection ELISA^(okys), Cat.No. 1,774,425, Boehringer Mannheim). The Boehringer Mannheim assay isbased on a sandwich-enzyme-immunoassay principle using mouse monoclonalantibodies directed against DNA and histones, respectively. This allowsthe specific determination of mono- and oligonucleosomes in thecytoplasmic fraction of cell lysates.

[0123] According to the vendor, apoptosis is measured in the followingfashion. The sample (cell-lysate) is placed into a streptavidin-coatedmicrotiter plate (“MTP”). Subsequently, a mixture of anti-histone-biotinand anti-DNA peroxidase conjugate are added and incubated for two hours.During the incubation period, the anti-histone antibody binds to thehistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA peroxidase antibody reacts with the DNAcomponent of the nucleosomes. After removal of unbound antibodies bywashing, the amount of nucleosomes is quantified by the peroxidaseretained in the immunocomplex. Peroxidase is determined photometricallywith ABTS7 (2,2′-Azido-[3-ethylbenzthiazolin-sulfonate]) as substrate.

[0124] Fold stimulation (FS=OD_(max)/OD_(veh)), an indicator ofapoptotic response, is determined for each compound tested at a givenconcentration. EC₅₀ values may also be determined by evaluating a seriesof concentrations of the test compound.

[0125] C. Experimental Results

[0126] 1. Introduction

[0127] Statistically significant increases of apoptosis (i.e., greaterthan 2 fold stimulation at a concentration of 100 μM) are furtherindicative that the cGMP-specific PDE inhibitor is useful in combinationwith a cisplatin derivative in the practice of this invention.Preferably, the EC₅₀ value for apoptotic activity should be less than100 μM for the compound to be further considered for potential use fortreating neoplastic lesions. EC₅₀ is herein defined as the concentrationthat causes 50% induction of apoptosis relative to vehicle treatment.

[0128] Apoptosis Assay

[0129] Reference compounds and test compounds were analyzed for theircGMP-specific PDE inhibitory activity in accordance with the protocolsfor the assay, supra. In accordance with those protocols, FIG. 11 showsthe effects of sulindac sulfide and exisulind on apoptotic and necroticcell death. HT-29 cells were treated for six days with the indicateddose of either sulindac sulfide or exisulind. Apoptotic and necroticcell death was determined as previously described (Duke and Cohen, In:Current Protocols in Immunology, 3.17.1-3.17.16, New York. John Wileyand Sons, 1992). The data show that both sulindac sulfide and exisulindare capable of causing apoptotic cell death without inducing necrosis.All data were collected from the same experiment.

[0130]FIG. 12 shows the effect of sulindac sulfide and exisulind ontumor growth inhibition and apoptosis induction as determined by DNAfragmentation. The top FIG. (12A) shows growth inhibition (open symbols,left axis) and DNA fragmentation (closed symbols, right axis) byexisulind. The bottom FIG. (12B) shows growth inhibition (open symbols)and DNA fragmentation (closed symbols) by sulindac sulfide. Growthinhibition was determined by the SRB assay after six days of treatment.DNA fragmentation was determined after 48 hours of treatment. All datawas collected from the same experiment.

[0131]FIG. 13 shows the apoptosis inducing properties of Compound E.HT-29 colon adenocarcinoma cells were treated with the indicatedconcentration of Compound E for 48 hours and apoptosis was determined bythe DNA fragmentation assay. The calculated EC₅₀ value was 0.05 μM.

[0132]FIG. 14 shows the apoptosis inducing properties of Compound B.HT-29 colon adenocarcinoma cells were treated with the indicatedconcentration of Compound B for 48 hours and apoptosis was determined bythe DNA fragmentation assay. The calculated EC₅₀ value was approximately175 μM. TABLE 6 Apoptosis Inducing Activity Among a Series of CompoundsReference compounds Fold induction at 100 μM Indomethacin <2.0 MY54454.7 Sulindac sulfide 7.9 Exisulind <2.0 E4021 <2.0 Zaprinast <2.0Sildenafil <2.0 EHNA <2.0 Test compounds Fold induction at 100 μM A <2.0B 3.4 C 5.6 D <2.0 E 4.6

[0133] In accordance with the fold induction protocol, suzra, CompoundsA through E were tested for apoptosis inducing activity, as reported inTable 6 above. Compounds B, C, and E showed significant apoptoticinducing activity, greater than 2.0 fold, at a dosage of 100 μM. Ofthese three compounds, at this dosage, only Compounds B and E did notinhibit COX but did inhibit cGMP-specific PDE.

[0134] The apoptosis inducing activity for a series of phosphodiesteraseinhibitors was determined. The data are shown in Table 7 below. HT-29cell were treated for 6 days with various inhibitors ofphosphodiesterase. Apoptosis and necrosis were determinedmorphologically after acridine orange and ethidium bromide labeling inaccordance with the assay described, supra. The data show cGMP-specificPDE inhibition represents a unique pathway to induce apoptosis inneoplastic cells. TABLE 7 Apoptosis Induction Data for PDE InhibitorsInhibitor Reported Selectivity % Apoptosis % Necrosis Vehicle 8 68-methoxy-IBMK PDE1 2 1 Milrinone PDE3 18 0 RO-20-1724 PDE4 11 2 MY5445PDE5 80 5 IBMX Non-selective 4 13

[0135] V. Mammary Gland Organ Culture Model Tests

[0136] A. Introduction

[0137] Test compounds identified by the above methods can be tested forantineoplastic activity by their ability to inhibit the incidence ofpreneoplastic lesions in a mammary gland organ culture system. Thismouse mammary gland organ culture technique has been successfully usedby other investigators to study the effects of known antineoplasticagents such as NSAIDs, retinoids, tamoxifen, selenium, and certainnatural products, and is useful for validation of the methods used toselect cGMP-specific PDE inhibitors useful in the present invention.

[0138] For example, female BALB/c mice can be treated with a combinationof estradiol and progesterone daily, in order to prime the glands to beresponsive to hormones in vitro. The animals are sacrificed and thoracicmammary glands are excised aseptically and incubated for ten days ingrowth media supplemented with insulin, prolactin, hydrocortisone, andaldosterone. DMBA (7,12-dimethylbenz(a)anthracene) is added to medium toinduce the formation of premalignant lesions. Fully developed glands arethen deprived of prolactin, hydrocortisone, and aldosterone, resultingin the regression of the glands but not the premalignant lesions.

[0139] The test compound is dissolved in DMSO and added to the culturemedia for the duration of the culture period. At the end of the cultureperiod, the glands are fixed in 10% formalin, stained with alum carmine,and mounted on glass slides. The extent of the area occupied by themammary lesions can be quantitated by projecting an image of the glandonto a digitation pad. The area covered by the gland is traced on thepad and considered as 100% of the area. The space covered by each of theunregressed structures is also outlined on the digitization pad andquantitated by the computer.

[0140] The incidence of forming mammary lesions is the ratio of theglands with mammary lesions to glands without lesions. The incidence ofmammary lesions in test compound treated glands is compared with that ofthe untreated glands.

[0141] B. Activity in Mammary Gland Organ Culture Model

[0142]FIG. 15 shows the inhibition of premalignant lesions in mammarygland organ culture by sulindac metabolites. Mammary gland organ cultureexperiments were performed as previously described (Mehta and Moon,Cancer Research, 46: 5832-5835, 1986). The results demonstrate thatsulindac sulfoxide and exisulind effectively inhibit the formation ofpremalignant lesions, while sulindac sulfide was inactive. The datasupport the hypothesis that cyclooxygenase inhibition is not necessaryfor the anti-neoplastic properties of desired compounds.

[0143] Conclusions Regarding Preferred PDE Inhibitors

[0144] To identify cGMP-inhibiting compounds that are useful fortreating neoplasia in combination with a cisplatin derivative, candidatecGMP-inhibiting compounds can be selected by testing them as describedabove.

[0145] Qualitative data of various test compounds and the severalprotocols are shown in Table 8 below. The data show that exisulind,sulindac sulfide, MY 5445, Compound B, and Compound E exhibit theappropriate activity to be used with a cisplatin derivative in thepractice of this invention. In addition, those same compounds (exceptfor sulindac sulfide and MY5445) are desirable because they lack COXinhibition activity. The activity of these compounds in the mammarygland organ culture validates the effectiveness of these compounds.TABLE 8 Activity Profile of Various Compounds Mammary Com- COX PDEGrowth Gland Organ pound Inhibition Inhibition Inhibition ApoptosisCulture Exisulind — ++ ++ ++ +++ Sulindac ++++ +++ +++ +++ — sulfideMY5445 ++++ +++ +++ +++ + A — — +++ ++ ++ B — +++ +++ +++ ++ D — — ++ —— E — +++− ++++ ++++ ++++ F — — ++ + — G — — +++ ++ +++ H — — ++ — —

[0146] Table 8. Code: Activity of compounds based on evaluating a seriesof experiments involving tests for maximal activity and potency.

[0147] −Not active

[0148] +Slightly active

[0149] ++Moderately active

[0150] +++Strongly active

[0151] ++++Highly active

[0152] Combination Treatment with a Cisplatin Derivative and a PDEInhibitor

[0153] The method of this invention involves treating a patient withneoplasia with both an antineoplastic platinum coordination complex anda cGMP-specific PDE inhibitor. There are a number of antineoplasticplatinum coordination complexes or cisplatin derivatives. In thisregard, the two terms are used interchangeably herein. Variousantineoplastic platinum coordination complexes (e.g.. cisplatin andcarboplatin) are disclosed. Other antineoplastic platinum coordinationcomplexes are disclosed in U.S. Pat. Nos. 4,996,337, 4,946,954,5,091,521, 5,434,256, 5,527,905, 5,633,243, all of which areincorporated herein by reference. Such compositions collectivelydisclose non-limiting examples of “antineoplastic platinum coordinationcomplexes” as that term is used herein.

[0154] This invention involves using combination therapy to treat apatient with neoplasia. By treating a patient with this combination ofpharmaceuticals, a cisplatin derivative and a cGMP-specific PDEinhibitor, therapeutic results can be achieved that are not seen witheither drug alone. As explained above, exisulind is one example of anappropriate cGMP-specific PDE inhibitor to be used in combination with acisplatin derivative in the practice of this invention. Exisulindinhibits both PDE5 and the new cGMP-PDE, and treatment of neoplasticcells with exisulind results in growth inhibition and apoptosis. (SeeTable 8).

[0155] Exisulind and the antineoplastic platinum coordination complex,carboplatin, were tested together to determine their combined effect onthe growth of a tumor cell line. The ability of the combination toinhibit tumor cell growth was tested by growing HT-29 cells in exisulind(FGN-1) doses from 25 μM-400 μM in the presence of carboplatin dosesvarying from 1 μM to 100 μM. (See FIGS. 15-20.) A standard SRB assay(see section III.A.) was performed to determine the drugs' effect oncell growth.

[0156] The data show the results on inhibition of cell growth aftertreatment with both exisulind and carboplatin. FIG. 16, for example,illustrates the effects on HT-29 cells grown in 25 μM exisulind andvarious doses of carboplatin. The percentage of growth inhibition withFGN-1 (exisulind) alone is shown in the first unshaded bar to the left,labeled FGN-1 alone. The effect on cells grown in 25 μM FGN-1 combinedwith 1 μM taxol is shown in the next bar to the right, labeled 1. Asillustrated in FIG. 16, the growth inhibition effects of combiningcarboplatin with a cGMP-specific PDE inhibitor, such as exisulind, aregreater than the effects of either treatment alone. This combinedbenefit is most pronounced at exisulind doses of 25 μM to 100 μM (FIGS.16-18) and at carboplatin concentrations of 10 μM or less.

[0157] The method of this invention involves using combination therapyto treat patients with neoplasia. Such combination therapy enhances thebenefit to the patient without increasing harmful side effects. Forexample, exisulind is one cGMP-specific PDE inhibitor that can be usedin combination with a cisplatin derivative in this invention.

[0158] Exisulind has no significant side effects when administered atits recommended dose of 300-400 mg/day. When administered at doseshigher than the recommended therapeutic levels, treatment with exisulindcan lead to elevated levels of liver enzymes. This effect is reversible,and liver enzymes return to normal levels when the administered dose ofexisulind returns to the traditionally recommended level or whentreatment is discontinued. The most serious side effects of cisplatinderivatives, on the other hand, are renal insufficiency andmyelosuppression. Since the side effects of the two drugs do notoverlap, a PDE inhibitor, such as exisulind, can be used in combinationwith a cisplatin derivative without increasing the harmful side effectsof the cisplatin derivative.

[0159] A cGMP-specific PDE inhibitor and a cisplatin derivative can beused in combination in at least two different ways. In the first method,the traditionally recommended dose range of the cisplatin derivative isreduced while its beneficial therapeutic effects are maintained and itsside effects are attenuated. The second method uses the traditionallyrecommended dose range of the cisplatin derivative with enhancedactivity but without increasing its side effects. In each of thesemethods, the patient is receiving both drugs, a PDE inhibitor and acisplatin derivative, either simultaneously or in succession.

[0160] The recommended dosage of a cisplatin derivative varies dependingon the type of cancer being treated and whether the cisplatin derivativeis being used in combination with another chemotherapeutic agent. In thepractice of this invention, a cGMP-specific PDE inhibitor is used as anadditional element of cancer treatment with a cisolatin derivative aloneor with a group of chemotherapeutic agents.

[0161] For the treatment of metastatic ovarian tumors, the typicalcisplatin dosage is 75 to 100 mg/M² once every four weeks when used incombination cyclophosphamide (Cytotoxan). Other chemotherapeutics usedin combination with cisplatin for ovarian tumors include paclitaxel,cyclophosphamide, or doxorubicin. As a single agent for the treatment ofmetastatic ovarian tumors, cisplatin is typically administered at a doseof 100 mg/M² once every four weeks.

[0162] For advanced bladder cancer, the recommended dose is 50 to 70mg/m² once every three to four weeks with cisplatin administered as asingle agent.

[0163] For the treatment of metastatic testicular tumors, therecommended cisplatin dose is 20 mg/m²m daily for five days when used incombination with another agent.

[0164] The typical dose of carboplatin, used for the treatment ofovarian cancer is 360 mg/m² administered once every 28 days.

[0165] In the practice of this invention, for each of the treatmentmethods mentioned above as well as other possible combinations,treatment with an appropriate cGMP-specific PDE inhibitor is added as anadditional element of the therapy. A cGMP-specific PDE inhibitor and anantineoplastic platinum coordination complex are used in combinationsuch that the blood levels of the inhibitor are at approximately theIC₅₀ value of the inhibitor for growth inhibition. In the case ofexisulind, it is recommended that the dose be about 200 to 400 mg/dayadministered between two to four times a day.

[0166] In one embodiment of this invention, the lower dose methodology,cisplatin is administered at a dosage lower than the traditionallyrecommended dose of 20, 50, or 75 mg/m² (for the indications above) ineach case, in combination with a cGMP-specific PDE inhibitor. Similarly,for carboplatin, a dosage less than 360 mg/m² in combination with acGMP-specific PDE inhibitor is practiced consistent with the lower dosemethodology of this invention. Accordingly, the combination of therapiesallows the benefits of antineoplastic platinum coordination complextreatment to be maintained while its side effects are reduced.

[0167] In the second embodiment, the dosage of cisplatin is maintainedat its traditionally recommended dose (e.g., at about 20 mg/m² daily, orbetween 50 to 70 mg/m² once every three to four weeks, or 75 to 100mg/m² once every four weeks, depending on the type of cancer beingtreated), and is administered in combination with a cGMP-specific PDEinhibitor. Similarly, for carboplatin, the current recommended dose(about 360 mg/m² administered once every 28 days) can be maintained incombination with a cGMP-specific PDE inhibitor. The combination, in thiscase, increases the efficacy of antineoplastic platinum coordinationcomplex treatment without increasing its harmful side effects.

[0168] In each of the aforementioned methodologies, the antineoplasticplatinum coordination complex and the cGMP-specific PDE inhibitor may beadministered simultaneously or in succession, one after the other.

[0169] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

We claim:
 1. A method of treating a patient with neoplastic cellscomprising causing cyclic GMP to increase in the neoplastic cells andexposing said neoplastic cells to a cisplatin derivative.
 2. The methodof claim 1 wherein the neoplastic cells are exposed to the cisplatinderivative at the same time cyclic GMP is increased.
 3. The method ofclaim 1 wherein the neoplastic cells are exposed to the cisplatinderivative before or after cyclic GMP is increased.